Membrane fusion, mediated by viral spike glycoproteins, is a key process in the infection cycle of all enveloped human and animal viruses. The overall goal of the proposed research is to understand the structural biology of viral membrane fusion. Although numerous crystal structures of the ectodomains of several enveloped virus spike glycoproteins have been determined over the years, we are only beginning to understand the structural transformations that take place when these proteins interact with membranes and how they drive membranes to fuse. In the previous grant period we have made substantial progress towards elucidating the structures of the fusion domains of influenza virus, human immunodeficiency virus, and Ebola virus in membrane environments. We have further been able to determine structural changes in these fusion domains that are conducive to membrane fusion. Most notably, membrane-bound influenza and Ebola virus fusion domains undergo large conformational changes in response to pH, i.e. there fusion trigger in the endosome. We also found that the conformation of the HIV fusion domain responds critically to cholesterol in the target membrane and that numerous critical mutants in these viral fusion domains affect their fusion function in a fashion that can be predicted by their altered structures. Based on these achievements, we now propose to test (1) the hypothesis that a deeper insertion of the Ebola virus fusion loop through a clenching of a fist motion is responsible for Ebola GP2-mediated membrane fusion, (2) the hypothesis that cholesterol and possibly cholesterol-rich lipid domains plays a key role in human immunodeficiency and other virus entry, and (3) the hypothesis that a common membrane perturbation mechanism involving lipid order and bilayer curvature changes underlies membrane fusion promoted by influenza, HIV and Ebola viruses.